Ultrasound‐mediated delivery of flexibility‐tunable polymer drug conjugates for treating glioblastoma

Abstract Effective chemotherapy delivery for glioblastoma multiforme (GBM) is limited by drug transport across the blood–brain barrier and poor efficacy of single agents. Polymer–drug conjugates can be used to deliver drug combinations with a ratiometric dosing. However, the behaviors and effectiveness of this system have never been well investigated in GBM models. Here, we report flexible conjugates of hyaluronic acid (HA) with camptothecin (CPT) and doxorubicin (DOX) delivered into the brain using focused ultrasound (FUS). In vitro toxicity assays reveal that DOX‐CPT exhibited synergistic action against GBM in a ratio‐dependent manner when delivered as HA conjugates. FUS is employed to improve penetration of DOX‐HA‐CPT conjugates into the brain in vivo in a murine GBM model. Small‐angle x‐ray scattering characterizations of the conjugates show that the DOX:CPT ratio affects the polymer chain flexibility. Conjugates with the highest flexibility yield the highest efficacy in treating mouse GBM in vivo. Our results demonstrate the association of FUS‐enhanced delivery of combination chemotherapy and the drug‐ratio‐dependent flexibility of the HA conjugates. Drug ratio in the polymer nanocomplex may thus be employed as a key factor to modulate FUS drug delivery efficiency via controlling the polymer flexibility. Our characterizations also highlight the significance of understanding the flexibility of drug carriers in ultrasound‐mediated drug delivery systems.

Our characterizations also highlight the significance of understanding the flexibility of drug carriers in ultrasound-mediated drug delivery systems. Among various experimental therapies, a topoisomerase (Top) II inhibitor, doxorubicin (DOX) or its liposomal form (Doxil), has been extensively investigated for the treatment of GBM. [2][3][4][5][6][7][8][9] Since the entry of intravenously injected therapies into the brain is limited by the blood-brain barrier (BBB) and the blood-tumor barrier (BTB), 10,11 focused ultrasound (FUS), a clinically viable tool, has been tested to transiently open the BBB and BTB. 11 While several preclinical reports demonstrate the efficacy of FUS-enhanced DOX delivery, the results have been variable, 9 and this strategy has not been used in humans except for one single study. 12 Another topoisomerase (I) inhibitor, irinotecan (Camptothecin-11 ) has also been successfully delivered to a rat GBM model after FUS-enabled BBB opening. However, the treatment did not achieve sufficient efficacy to improve the survival of F98 glioma model. 13 Similar concerns of the lack of efficacy in single agent chemotherapies have been raised in treating various other types of cancer.
To address these issues, we 14 and others 15 have demonstrated that a combination of two Top inhibitors, DOX and CPT, exhibits a highly synergistic activity against various cancers including brain tumors. 16,17 CPT and DOX can inhibit both Top I and II enzymes that regulate DNA transcription and cell replication, and this combination strategy could potentially offer a higher efficacy than a single agent therapy. Studies have indicated increased levels of Top I enzymes in glioma cells in contrast to the normal brain tissue, [18][19][20][21][22] suggesting a selective activity of CPT in brain cancer. 18 Previous works also suggest that the DOX-CPT ratios in the drug combination would affect the treatment efficacy. 14,23 However, success in translating the combination of DOX-CPT into the clinic has been hindered primarily due to solubility issues associated with CPT and the ability to deliver synergistic DOX-CPT ratios to the target site. We address both issues by conjugating CPT and DOX to HA, thereby improving the solubility of CPT and preserving the drug ratios. In addition, FUS enables spatially targeted delivery of these precise drug ratios to the tumor, ensuring synergistic antitumor activity of CPT and DOX at the site of action.
As we use FUS for physically enhancing drug transport across the BBB, it is also vital to understand the association between the delivery efficiency and the physical characteristics of the HA conjugates, which can be affected by the DOX-CPT drug ratio. While previous reports using FUS for drug delivery to the brain only demonstrated that the size of the drug/agent could affect the delivery under the same ultrasound parameters, 24,25 other physical parameters of the drug carrier can also play an important role and have not been explored. Additionally, previous works did not study this relationship using polymer conjugates. We address these issues by using small-angle x-ray scattering (SAXS) characterization of the conjugates to decipher the relationships among DOX:CPT ratio, the polymer chain flexibility, and the drug delivery efficiency, and efficacy showed in the animal studies.
Here, we evaluated HA conjugates of DOX and CPT for the treatment of GBM. To co-deliver these two drugs at specific ratios, we used HA as the drug carrier 14 for its biocompatibility and the targeting specificity for CD44, which is over-expressed on human GBM cells. 26 Based on an in vitro cell toxicity assay, in vivo drug delivery, and treatment efficacy evaluation in a murine GBM model GL261, we identified a synergistic combination of Top I (CPT) and Top II inhibitors (DOX) at specific ratios in the HA conjugates. Leveraging previous work demonstrating the ability of FUS to enhance chemotherapeutic delivery into the GBM, [3][4][5][6][7][8]27 we demonstrate that FUS enables the delivery of HA-CPT-DOX conjugates into the brain for the treatment of GBM. We further show that SAXS characterizations of the conjugates reveal that DOX:CPT ratio affects the polymer chain flexibility.
Conjugates with the highest flexibility (lowest stiffness) yielded the highest efficacy in treating mouse glioblastoma in vivo after FUS BBB opening.

| Synthesis and characterization of hyaluronic acid conjugates
DOX and CPT were chemically conjugated to HA forming single drug conjugates or the dual drug conjugates at 3 molar ratios (HA-DOX-CPT at R2, R5 and R15, R = molar ratio of DOX:CPT in the conjugate) (Table S1). The amount of each drug conjugated to HA was measured using the fluorescence spectra that was specific for each molecule (Table S2). Fourier transform infrared spectroscopy (FTIR) confirmed the formation of amide and ester bonds suggesting the successful covalent conjugation of CPT and DOX to HA (Figure 1a). The 50-kDa HA was conjugated to the hydrophobic drugs CPT and DOX. The covalent linkage to HA was validated using FTIR, as shown in Figure 1. For HA, characteristic peaks are observed due to the stretching vibration of OH and NH groups (3309 cm À1 ) (a), and the symmetric and asymmetric vibration of COO (1613 cm À1 ) (b) and 1400 cm À1 (c), respectively. Furthermore, the characteristic peak at 1030 cm À1 (d) is attributed to the C O C hemiacetalic saccharide linkages in the polymeric chain. 28 29 FTIR spectrum of free CPT shows absorp- (r) represents contribution from the adjacent hetero-aromatic nuclei. 30 The FTIR spectrum for HA-DOX-CPT suggested that the polysaccharide structure of HA backbone was intact. While the spectrum also confirms the presence of DOX through its strong characteristic peaks  Figure S1 and Table S3) and nanoparticle tracking analyses (NTA) (Figure 1c). TEM revealed a micellar appearance, suggesting that drug conjugation imparts hydrophobicity to HA resulting in the self-assembly. NTA revealed the average mean size to be 151. 5

| Cell viability study
To investigate the antitumor effects and optimize the drug ratio in the

| FUS-triggered drug delivery
We next tested whether the candidate DOX-HA-CPT (R2 and R15), which showed synergistic antitumor effects in the cell viability study, can be delivered across the BBB in healthy mouse brains.
Following one session of FUS-enabled BBB opening together with DOX-HA-CPT intravenous administration (5 mg/kg of body weight, FUS started immediately after drug administration), wild-type mice were sacrificed, and brain samples were harvested for fluorescence microscopic assessment. The R15 conjugates were delivered to the striatum areas (Figure 3a), even with limited plasma drug concentrations at the time of sacrificing the animals (<5% injected dose at 2 h post drug administration, see Figure S3). The delivery of both DOX and CPT was significantly enhanced post FUS treatment ( Figure 3b).
However, surprisingly, the enhancement of FUS-triggered delivery of R2 was not as evident as that of R15 (Figure 3c). FUS-treated areas exhibited moderately elevated delivery of R2, but the fluorescent intensity was not significantly higher compared to that of non-FUS-treated areas ( Figure 3d). As R2 was delivered far less than R15, we cannot find evident hyperintense spots on the similar planes (which typically should be in the middle of the dorsal-ventral axis of the brain, as shown in Figure 3a) in the R2 group. The R2 delivery was mostly situated closer to the cortex regions due to a higher vascular density.
Of note, the two DOX-HA-CPT tested here contained the same amount of DOX, but R2 carries 7.5 times more CPT than R15. However, after FUS-enabled BBB opening, R15 was able to deliver more of both chemotherapeutics compared to R2. Considering the same FUS settings were used in the two groups, we speculated that the DOX:CPT ratio may affect some properties of the DOX-HA-CPT relevant for FUS drug delivery efficiency. As shown in Figure

| Treatment efficacy
To test whether HA-CPT-DOX can be used for treating GBM, a survival study was performed in the GL261 model assessing the treatment efficacy of R2 and R15 with or without the FUS treatment ( Figure 4a).
After intracranial tumor inoculation, we monitored the tumor growth  (Table 1). FUS-R15 treatment resulted in the longest median survival and the most number of mid-term survivors among all the groups. Overall, R15 therapies (monotherapy or with FUS) were more efficacious than R2 therapies. This is in agreement with the conclusion of the cell viability assays based on the IC50 and C.I assessments (Table 2). Additionally, this in vivo efficacy result is consistent with the observation that more CPT and DOX was delivered via R15 than R2 after FUS-enabled BBB opening ( Figure 3).
To further test whether FUS can enhance the delivery of chemotherapies locally to tumors, we performed immunohistochemistry In addition, we stained CD3 and CD8 in the brain sections to assess the T-cell infiltration. We found that FUS significantly enhanced

| Polymer flexibility
As described earlier, it was surprising to find R2, which was effective in the cell assay, had limited delivery after FUS-triggered BBB opening compared to R15. To further address this question and link other physical properties of the polymers to the FUS-trigged treatment outcomes, we used SAXS to characterize the polymer conformational properties.
SAXS was used to determine the equilibrium rigidity of DOX-HA-CPT in solution. 31 The polymer equilibrium flexibility/rigidity can be described by the Kuhn segment length (Figure 6a). 32 A strong scattering intensity observed for DOX-HA-CPT confirmed its particulate shape as seen with TEM ( Figure 1b) and AFM (supporting materials S1). To evaluate the Kuhn segment length, the SAXS data were fit to a model of a "Worm-Like Chain" 33 describing the conformation of semi-rigid and rigid macromolecules (Figure 6b). This model was chosen due to the charged nature of polymers in this study.
In addition, the form-factors of aggregates and the hard sphere repulsion structure factor were applied to describe the interparticle interactions. Several structural parameters were extracted from the fitting including the values of Kuhn segment length and radius of hard sphere repulsion (RHS, Figure 6a). 32 A clear correlation (Figure 6c) was observed between the RHS and Kuhn segment length as a function of drug ratio, R. Polymer conjugates with a higher ratio of DOX to CPT exhibit a lower Kuhn segment length and higher RHS.
This suggests a more drastic morphological transition on polymeric chain with an increasing R. Therefore, in this study, the SAXS results demonstrated that R15 and R5 are flexible in nature, whereas R2 is a semi-flexible polymer that is more rigid than R15 and R5.

| DISCUSSION
In this study, CPT and DOX were delivered at specific ratios as DOX-HA-CPT conjugates after FUS-enabled blood-brain/tumor barrier disruption for treating GBM in mice. We identified that DOX-CPT synergy was associated with DOX:CPT ratio in the HA-based nanoconjugates in vitro. In addition, another ratiometric dependency was also observed

| CONCLUSION
Taken together, we have developed an FUS-enhanced HA-based platform to deliver combination chemotherapies to treat GBM. By optimizing the drug ratio in the dual-drug-carrying nanoconjugates, this platform was able to change the polymer flexibility, achieve effective drug delivery, and improve survival benefits in a mouse GBM model. Our results also suggest that the effectiveness of FUS-mediated brain delivery may be dependent on the physical properties of nanocomplexes such as the flexibility, which has not been identified in previous reports.  Table S1. At the end of all the reactions, the products were purified with Sephadex G-25 PD-10 desalting columns (5000 M.W. exclusion limit) followed by overnight dialysis (3500 MWCO) against DI water.
The dialyzed product was then lyophilized and stored at 4 C, prior to reconstituting with PBS for subsequent in vitro or in vivo studies. The amounts of DOX and CPT incorporated on HA were assessed using fluorescence: Ex/Em 470/590 for DOX and 370/448 nm for CPT-for each molecule, respectively.

| FTIR characterization
Infrared spectra were collected with NicoletTM FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA) within range of 600-4000 cm À1 and 4 cm À1 spectral resolution. Lyophilized controls and DOX-HA-CPT conjugates were individually placed on the diamond surface of the ATR device and 32-scan interferogram was recorded for each sample. Thermo ScientificTM OMNICTM Spectra software was used to analyze the peaks, after applying baseline, atmospheric and ATR corrections on the raw spectra.

| Size measurements
The Malvern Panalytical NanoSight NS300 Instrument is an analytical sizing instrument. This instrument utilizes NTA to accurately characterize non-normally size distributed particles by measuring the diffusion coefficient of each particle individually. To ensure that the instrument was sizing particles properly, 100 nm polystyrene-latex microsphere Due to low concentration of nanoparticles, we assume the structure factor S(q) = 1 (interparticle interactions are neglected). The scattered intensity curves were fitted using SASFit software. 47 • Polydisperse hard sphere model The following form was used to describe the spherical shell form factor, where R is the radius of sphere and ΔSLD is the difference in scattering length densities (SLD) between particle and solvent.
Taking nanoparticles polydispersity into account, a Schulz-Zimm distribution of R with polydispersity parameter σ was included in the following way: where Z ¼ 1 σ 2 À 1. Since the polydispersity parameter σ and radius of sphere are correlated parameters, the σ value was set to 0.3 for all fitting procedures.
• Generalized Gaussian coil model The following form factor of Generalized Gaussian coil was used: The fitting parameters for this model are R g (gyration radius) and ν (Flory exponent).
• Worm-like chain model: The form factor of a worm-like chain with contour length L, Kuhn length A, and diameter d has been described previously. 33

| In vivo imaging
The MRI and BLI were performed in the Brigham and Women's Hospital Research Imaging Core/Small Animal Imaging Lab (SAIL).
T1-weighted and T2-weighted MR imaging were acquired using a

| Fluorescent imaging and immunohistochemistry
Fluorescent imaging of brain sections was acquired using a fluorescence stereo zoom microscope (ZEISS Axio Zoom.V16, Carl Zeiss AG, Oberkochen, Germany). Transcardiac perfusion was performed before harvesting the brains for drug delivery assessment ( Figure 3) and the histology study ( Figure 5). For assessing FUS-trigged drug delivery, mice were sacrificed 2 h after the treatment, and the harvested brains were fixed in 4% PFA for 24 h followed by 30% sucrose solution bath for cryoprotection. Brains were embedded in OCT before cryosectioning (with a thickness of 80 μm). Serial sectioning started from the dorsal surface of the brain all the way to the ventral surface. Sections were spaced with a gap of 480 μm (every six sections). A total of nine sections per brain were used for quantification and we measured the total fluorescent intensity of all the sections for each mouse.
Immunohistochemistry was performed on the paraffin-embedded brains on the Leica Bond III automated staining platform using the

| Statistical analysis
All statistical analyses were carried out using Prism Graphpad 9.2 software. All data are presented as mean ± SEM (standard error of mean) unless specified, student's t test or one-way ANOVA with Tukey's HSD analysis were used to determine significance. p values represent levels of significance (***p < 0.001, **p < 0.01, and *p < 0.05).